Stereoselective synthesis of [13C]methyl 2-[15N]amino-2-deoxy-beta-D-glucopyranoside derivatives.

Article abstract of DOI:10.1016/S0008-6215(01)00203-8

Efficient syntheses of three [13C]methyl 2-[15N]amino-2-deoxy-beta-D-glucopyranoside derivatives are described. Amination of the D-glucal with (saltmen)Mn(15N) proceeded with 11:1 stereoselectivity favoring the gluco configuration; subsequent methylation of the [15N]lactol using [13C]iodomethane and silver(I) oxide afforded the doubly labeled beta glucoside in high yield. This compound served as the common precursor for three [13C]methyl 2-[15N]aminoglucosides: (2-[15N]trifluoroacetyl-), (2-[15N]acetyl-), and (2-[15N]azido-). Selected heteronuclear coupling constants are reported.

Full text of DOI:10.1016/S0008-6215(01)00203-8

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Carbohydrate Research 334 (2001) 271–279
Stereoselective synthesis of [13C]methyl
2-[15N]amino-2-deoxy-b-
D-glucopyranoside derivatives
Fabien P. Boulineau, Alexander Wei*
Department of Chemistry, Purdue Uni6ersity, 1393 Brown Building, West Lafayette, IN 47907-1393, USA
Received 1 May 2001; accepted 9 July 2001
Abstract
Efficient syntheses of three [13C]methyl 2-[15N]amino-2-deoxy-b-
D
-glucopyranoside derivatives are described.
Amination of the D
-glucal with (saltmen)Mn(15N) proceeded with 11:1 stereoselectivity favoring the gluco configura-
tion; subsequent methylation of the [15N]lactol using [13C]iodomethane and silver(I) oxide afforded the doubly labeled
b glucoside in high yield. This compound served as the common precursor for three [13C]methyl 2-
[15N]aminoglucosides: (2-[15N]trifluoroacetyl-), (2-[15N]acetyl-), and (2-[15N]azido-). Selected heteronuclear coupling
constants are reported. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Stereoselectivity; 13C-Labeling; 15N-Labeling; Heteronuclear coupling constants
1. Introduction
require additional steps for their use as syn-
15
thetic intermediates. Stereoselective N incor-
Isotopically labeled carbohydrates are valu-
able compounds for conformational studies by
nuclear magnetic resonance (NMR) spec-
troscopy and for the elucidation of biosyn-
thetic pathways of complex oligosacchar-
ides.1,2 Numerous synthetic and biosynthetic
routes have been developed for deuterated and
13C-labeled monosaccharide derivatives, many
of which are commercially available.† In con-
trast, 15N-labeled aminosaccharides are much
less accessible. Although synthetic approaches
poration into protected monosaccharide
derivatives at a late stage is much more effi-
cient and is especially relevant for multistep
synthetic sequences. Methods to consider for
stereoselectively introducing nitrogen into car-
bohydrate derivatives via electrophilic addi-
tion to glycals have been developed, including
nitrochlorination,4 azidonitration,5 phospho-
ramidoglycosylation,6 [4+2] cycloaddition,7
sulfonamidoglycosylation,8
amination
by
Mn(V) nitrido complexes,9 and acetamidogly-
to 2-[15N]-
-glucosamine derivatives have
D
cosylation.10
been described,3 the yields are low and would
We present an efficient and stereoselective
route toward doubly labeled [13C]methyl 2-
[15N]amino - 2 - deoxy - b -
D
- glucopyranoside
derivatives with different nitrogen-containing
functional groups at C-2. We applied the salt-
men(Mn)(V)nitrido methodology developed
by Du Bois et al.9 to the stereoselective
[15N]amination of glycals (Fig. 1) for the fol-
lowing reasons: (i) the amination was reported
* Corresponding author. Tel.: +1-765-4945257; fax: +1-
765-4940239.
E-mail address: alexwei@purdue.edu (A. Wei).
† Leading suppliers include Omicron Biochemicals, Inc.
(http://www.omicronbio.com) and Sigma–Aldrich Chemicals
(http://www.sigma-aldrich.com).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00203-8

272
F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
2. Results and discussion
to proceed with good facioselectivity favoring
the gluco configuration; (ii) the amination
conditions were compatible with several dif-
ferent protecting groups, including the elec-
tron-rich and acid-sensitive p-methoxy-
benzylidene acetal; and (iii) the (saltmen)-
Mn(15N) reagent could be easily prepared on a
gram scale from aqueous 15NH4OH, a rela-
tively inexpensive source of 15N. An efficient
synthetic route would enable us to produce
sufficient quantities of C, N-labeled carbo-
hydrates for solid-state NMR studies such as
rotational-echo double resonance (REDOR)
spectroscopy.11
We first optimized the preparation of the
D
-glucal 4 as a precursor for [15N]amination
(Scheme 1). Treatment of the known phenyl
4,6 - O - p - methoxybenzylidene - 1 - thio - b -
glucopyranoside (1)12 with 2-methoxypropene
in the presence of catalytic amounts of -cam-
D
-
D
phorsulfonic acid afforded the crystalline
derivative 2 in 90% yield. This compound was
treated with an excess of lithium naph-
thalenide at −78 °C13 to give the crystalline
glycal derivative 3 in 75% yield, which was
then protected as the tert-butyldimethylsilyl
ether derivative 4 in 97% yield.
13
15
Stereoselective amination9 of glucal 4 was
achieved by the slow addition of a stoichio-
metric amount of (saltmen)Mn(N) in
dichloromethane to a concentrated solution of
the -glucal and trifluoroacetic anhydride (3.5
D
equiv) in dichloromethane at room tempera-
ture, yielding lactol 5 as a diastereomeric mix-
ture in 66% yield (Scheme 2). The optimized
reaction conditions and determination of
diastereomeric ratios of the aminated product
were accomplished using natural-abundance
(saltmen)Mn(N). Acetylation of the lactols
and careful separation by silica-gel chro-
Fig. 1. 15N amination of glycals using the (saltmen)Mn(N)
methodology developed by Du Bois et al.9
matography enabled us to evaluate their
D
-
1
gluco/ -manno configurations by H NMR
D
coupling constant analysis. Two fractions
were isolated, one of them being the enantiop-
ure 1-O-acetyl-a- -glucosamine derivative 6
D
(76%) and the other a mixture of the remain-
ing three diastereomers (18%), the majority
Scheme 1. Reagents and conditions: (a) 2-methoxypropene,
CSA, THF, 40 °C (90%); (b) lithium naphthalenide, THF,
−78 °C (75%); (c) TBSCl, imidazole, DMF, 23 °C (97%).
PMP=p-methoxyphenyl, TBS=tert-butyldimethylsilyl.
being the 1-O-acetyl-b-
D
-glucosamine deriva-
tive. The -gluco/ -manno ratio was greater
D
D
than 11:1 based on the yields and the integra-
tion values of the H-1 peaks. The 4,6-p-
methoxybenzylidene acetal was determined to
be important for the high stereoselectivity at
C-2: amination of tribenzyl- -glucal 7 resulted
D
in an approximately 1:1 gluco/manno ratio,
suggesting that the p-methoxybenzylidene ace-
tal stabilizes a transition state favoring the
gluco configuration by restricting the confor-
mation of glucal 4.
The 15N-labeled lactol 5 was treated with
silver(I) oxide and [13C]iodomethane in tetra-
hydrofuran at room temperature to afford the
doubly 13C, 15N-labeled methyl glycoside 8 in
Scheme 2. Reagents and conditions: (a) (saltmen)Mn(N),
(CF3CO)2O, CH2Cl2, 23 °C (66%); (b) Ac2O, pyridine, 23 °C
(94% overall yield, 76% isolated yield of 6). PMP=p-
methoxyphenyl, TBS=tert-butyldimethylsilyl.

F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
273
Scheme 3. Reagents and conditions: (a) Ag2O, 13CH3I, THF, 23 °C (76% isolated yield); (b) (i) TBAF, THF, 23 °C; (ii)
AcOH–THF–water, 45 °C (94% after two steps); (c) (i) NaOH, CH3OH–water, reflux; (ii) Ac2O, CH3OH, 0 °C; (iii) AcOH–
THF–water, 45 °C (81% after three steps); (d) (i) NaOH, CH3OH–water, reflux; (ii) TfN3, K2CO3, CuSO4 (cat), 90% CH3OH,
0–23 °C (73% after three steps). PMP=p-methoxyphenyl, TBS=tert-butyldimethylsilyl.
76% isolated yield (Scheme 3). Deprotection
of 8 by fluoride treatment and hydrolysis un-
der mildly acidic conditions produced
observed on the 15N NMR spectrum (50
MHz) at 70.7 ppm, demonstrating that no
isotopic scrambling occurred during the azide
formation. It is also noteworthy that the
2JN,H2 coupling constant was either unde-
tectable or very small (B1 Hz) for com-
pounds 9–11, whereas 3JN,H3 coupling
constants (1.5–2.0 Hz) were observed in the
case of compounds 9 and 10. 15N–13C cou-
[13C]methyl
oxy-b-
2-[15N]trifluoroacetamido-2-de-
D
-glucopyranoside (9) as a white solid
in 94% yield over two steps. Compound 8
could also be cleanly hydrolyzed by sodium
hydroxide in aqueous methanol to the free
amine, then acetylated and deprotected as de-
scribed above to afford [13C]methyl 2-
1
pling constants were also evaluated: JN,C2 for
[15N]acetamido-2-deoxy-b-
D
-glucopyranoside
the azido compound 11 (3.2 Hz) was only a
third that of the amido compounds 9 and 10
(10) as a white solid in 81% yield over three
steps. Treating the free amine with freshly
prepared triflyl azide (TfN3) and catalytic
quantities of copper(II) sulfate14 produced a
2
(10.2–10.6 Hz), whereas JN,C1 was 3.5 Hz for
compound 11 but undetectable for the other
two compounds.
2-azido-2-deoxyglucose
upon deprotection afforded [13C]methyl 2-
[15N]azido-2-deoxy-b-
-glucopyranoside (11)
derivative,
which
In summary, we have developed an expedi-
tious and stereoselective route to [13C]methyl
D
2-[15N]amino-2-deoxy-b-
-glucopyranoside
D
as a white crystalline solid in 73% yield after
three steps. It is worth mentioning that the
rate of azide formation was significantly accel-
erated by increasing the concentration of wa-
ter in the reaction mixture; reactions
conducted in 10:9:1 CH2Cl2 –CH3OH–water
were complete within 14 h, whereas reaction
mixtures containing 1% water or less required
several days to reach completion.
derivatives from readily available starting ma-
terials and relatively inexpensive sources of
15N and 13C. The 4,6-p-methoxybenzylidene
acetal plays
a
key role in the high
gluco:manno stereoselectivity of the 15N ami-
nation step. Further solution and solid-state
NMR studies of these compounds are in pro-
gress and will be reported in due course.
1
The H NMR spectrum of compound 8
(300 MHz) in CDCl3 confirms double isotope
incorporation (Fig. 2). Interestingly, all four
doubly labeled compounds 8–11 exhibited an
3. Experimental
General methods.—Melting points were de-
termined with a capillary tube melting point
apparatus and are uncorrected. IR spectra
were acquired with a Perkin–Elmer Spectrum
3
identical JC,H coupling constant across the
glycosidic bond (J 4.5 Hz in each case). In the
case of compound 11, one single peak was

274
F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
Phenyl
2,3-di-O-isopropylidene-4,6-O-p-
- glucopyran-
2000 FTIR spectrometer. Optical rotations
were measured at 20–25 °C with a Rudolph
Research AUTOPOL® III polarimeter. 1H
and 13C spectra were recorded on a Varian
Unity Inova NMR spectrometer operating at
300 and 75 MHz, respectively, or a Bruker
DRX 500 operating at 500 and 125 MHz,
respectively. The spectra were referenced to
the solvent used: (7.27 and 77.23 ppm for
CDCl3, 3.31 and 49.15 ppm for CD3OD, 7.16
and 128.39 ppm for C6D6), unless otherwise
methoxybenzylidene - 1 - thio - i -
D
oside (2).—2-Methoxypropene (5.5 mL, 57
mmol) was added to a solution of 1 (4.48 g,
11.4 mmol) and
D
-camphorsulfonic acid (133
mg, 0.6 mmol) in dry THF (100 mL), and the
mixture was stirred under Ar in the dark at
40 °C for 1 h. The mixture was cooled to 0 °C,
quenched with satd aq NaHCO3 (30 mL),
extracted with EtOAc (150 mL), dried over
Na2SO4, and concentrated. The residue was
crystallized from 95% hot EtOH (150 mL) to
give 2 as a white crystalline solid (4.46 g,
90%): mp 145–148 °C; [h]D −47° (c 1,
CHCl3); IR (thin film): 2986, 2893, 1615, 1518,
1382, 1249, 1099, 1067, 1041, 963, 831, 751
15
stated. N spectra were recorded on a Bruker
DRX 500 operating at 50 MHz and refer-
enced to an external 1 M solution of 15N-la-
15
beled urea in Me2SO at 77 ppm (liquid NH3
is referenced to 0 ppm). Mass spectra were
acquired using either a Hewlett–Packard
5989B or a Finnigan 4000 mass spectrometer.
The purity of the products was assessed using
TLC on Silica Gel 60 F254 (E. Merck) with
either ethanolic p-anisaldehyde or ninhydrin
staining solutions. Chromatographic separa-
tions were performed using silica gel (ICN
SiliTech, 32–63 mm). Elemental analyses were
performed in the Department of Chemistry,
Purdue University.
1
cm−1; H NMR (C6D6): l 7.64 (m, 2 H,
ArꢀH), 7.52 (m, 2 H, JA,A%=JX;X% 2.1, JA,X
=
JA%,X% 8.7 Hz, ArꢀH), 7.03 (m, 3 H, ArꢀH),
6.79 (m, 2 H, JA,A%=JX;X% 2.1, JA,X=JA%,X% 8.7
Hz, ArꢀH), 5.20 (s, 1 H, CHꢀPMP), 4.57 (d, 1
H, J1,2 9.6 Hz, H-1), 4.14 (dd, 1 H, J5,6a 4.8,
J6a,6b 10.2 Hz, H-6a), 3.65 (dd, 1 H, J3,4 9.6,
J4,5 8.7 Hz, H-4), 3.49 (t, 1 H, J5,6b 10.2 Hz,
H-6b), 3.44 (dd, 1 H, J2,3 8.7 Hz, H-2), 3.35
(dd, 1 H, H-3), 3.24 (s, 3 H, OCH3), 3.07
(ddd, 1 H, H-5), 1.37 (s, 3 H, CH3), 1.35 (s, 3
Fig. 2. Partial 1H NMR (300 MHz, CDCl3) of compound 8 which served as the common precursor for the synthesis of the doubly
labeled compounds 9–11.

F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
275
H, CH3); 13C NMR (C6D6): l 160.93 (1 C,
CꢀOCH3 Ar), 134.51, 132.51, 130.74, 129.32,
128.78, 128.38, 114.10 (11 C, C Ar), 111.52,
101.70 (2 C, C acetal), 85.78 (1 C, C-1), 80.55,
79.71, 77.09, 72.58, 68.98 (5 C, C-2,-3,-4,-5,-6),
55.09 (1 C, OCH3), 27.15, 26.99 (2 C,
C(CH3)2); CIMS: m/z 431, [M+1]+. Anal.
Calcd for C23H26O6S: C, 64.17; H, 6.09; S,
7.45. Found: C, 64.03; H, 6.08; S, 7.21.
mmol) and tert-butylchlorodimethylsilane
(731 mg, 4.8 mmol). After 18 h, satd aq
NaHCO3 (20 mL) was added at 0 °C along
with Et2O (50 mL) and water (10 mL). The
mixture was extracted, the organic phase
washed with water (3×10 mL), dried over
Na2SO4, and concentrated. The crude residue
was eluted from a plug of silica gel with 9:1
hexanes–EtOAc containing 0.1% of Et3N to
give 4 as a clear syrup (603 mg, 97%): [h]D
−59.5° (c 2, CHCl3); IR (thin film): 2891,
2954, 2930, 2857, 1640, 1615, 1518, 1381,
1251, 1233, 1170, 1128, 1105, 1073, 1036,
4,6-O-p-Methoxybenzylidene- -glucal (3).
D
—The 1 M lithium naphthalenide solution in
THF was prepared according to the literature
protocol.13 To a solution of 2 (1.0 g, 2.3
mmol) in dry THF (30 mL) under Ar and at
−78 °C was added the freshly prepared
lithium naphthalenide solution in THF (10
mL, 9.9 mmol) dropwise for 30 min, and the
mixture was stirred until complete disappear-
ance of the starting material by TLC (2:1
hexanes–EtOAc). The reaction was quenched
with 4:1 THF–AcOH (2 mL). The flask was
removed from the −78 °C bath and diluted
with CH2Cl2 (80 mL) and aq 0.5 M NaOH (10
mL). The reaction mixture was separated,
dried over Na2SO4 and concentrated. The re-
sulting solid residue was crystallized from 9:1
hexanes–EtOAc to afford a white crystalline
solid (462 mg, 75%): mp 133–134 °C; [h]D
−19° (c 1, CHCl3); IR (thin film): 3285, 2874,
1639, 1615, 1519, 1377, 1253, 1233, 1172,
1
1017, 1007, 867, 836, 824, 779 cm−1; H
NMR (CDCl3): l 7.43 (m, 2 H, JA,A%=JX;X%
2.7, JA,X=JA%,X% 8.7 Hz, ArꢀH), 6.90 (m, 2 H,
JA,A%=JX;X% 2.7, JA,X=JA%,X% 8.7 Hz, ArꢀH),
6.30 (dd, 1 H, J1,2 6.0, J1,3 1.5 Hz, H-1), 5.56
(s, 1 H, CHꢀPMP), 4.67 (dd, J2,3 2.1 Hz, H-2),
4.50 (ddd, 1 H, J3,4 7.2 Hz, H-3), 4.34 (dd, 1
H, J5,6a 4.2, J6a,6b 9.6 Hz, H-6a), 3.93–3.79 (m,
6 H, H-4,-5,-6b, OCH3), 0.94 (s, 9 H,
C(CH3)3), 0.14 (s, 3 H, SiCH3), 0.13 (s, 3 H,
SiCH3); 13C NMR (CDCl3): 160.12 (1 C,
CꢀOCH3), 143.51 (1 C, C-1), 130.07, 127.51,
113.67 (5 C, C Ar), 105.62 (1 C, C acetal),
101.44, 80.74, 68.99, 68.50, 67.50 (5 C, C-2,-3,-
4,-5,-6), 55.46 (1 C, OCH3), 26.01 (3 C,
C(CH3)3), 18.45 (1 C, C(CH3)3), −4.17 (1 C,
SiCH3), −4.60 (1 C, SiCH3); CIMS: m/z 379,
[M+1]+. Anal. Calcd for C20H30O5Si: C,
63.46; H, 7.99; Si, 7.42. Found: C, 63.13; H,
7.88; Si, 7.33.
1
1125, 1096, 1069, 1032, 1006, 823 cm−1; H
NMR (CDCl3): l 7.44 (m, 2 H, JA,A%=JX;X%
2.7, JA,X=JA%,X% 8.4 Hz, ArꢀH), 6.91 (m, 2 H,
JA,A%=JX;X% 2.1, JA,X=JA%,X% 8.7 Hz, ArꢀH),
6.35 (dd, 1 H, J1,2 6.3, J1,3 1.8 Hz, H-1), 5.57
(s, 1 H, CHꢀPMP), 4.79 (dd, J2,3 2.1 Hz, H-2),
4.52 (m, 1 H, J3,OH 4.2, J3,4 6.3 Hz, H-3), 4.36
(dd, 1 H, J5,6a 4.5, J6a,6b 9.9 Hz, H-6a), 3.95
(dd, 1 H, J4,5 10.5 Hz, H-4), 3.85–3.77 (m, 5
H, H-5,-6b,OCH3), 2.18 (d, 1 H, J3,OH 4.2 Hz,
OH); 13C NMR (CDCl3): l 160.47 (1 C,
CꢀOCH3 Ar), 144.30 (1 C, C-1), 129.72,
127.77, 113.93 (5 C, C Ar), 103.78 (C acetal),
101.96, 80.86, 68.54, 68.49, 66.77 (5 C, C-2,-3,-
4,-5,-6), 55.51 (1 C, OCH3); CIMS: m/z 265,
[M+1]+. Anal. Calcd for C14H16O5: C, 63.63;
H, 6.10. Found: C, 63.52; H, 6.15.
Preparation of (saltmen)Mn(15N).—To a
stirring solution of H2saltmen15 (1.3 g, 4
mmol) in CH3OH (50 mL) at 50 °C was added
Mn(OAc)2·4 H2O (1.03 g, 4 mmol) portion-
wise. The mixture was then brought to reflux
for 1 h, cooled to 23 °C, and stirred for 30
min. To the vigorously stirring dark mixture
15
was added aq NH4OH (6 M, 5 g, 30 mmol),
followed by the dropwise addition of commer-
cial bleach (ꢀ0.7 M NaOCl, 18 mL, 12
mmol) for 45 min. The heterogeneous mixture
was cooled to 0 °C and diluted with CH2Cl2
(150 mL). The organic phase was abundantly
washed with small portions of water (4×50
mL), concentrated, and passed through a
Brockmann activity IV basic Al2O3 plug with
CH2Cl2 (200 mL). The dark-green solution
was concentrated and crystallized from 2:1
hexanes–EtOAc to afford a lustrous dark
3-O-tert-Butyldimethylsilyl-4,6-O-p-meth-
oxybenzylidene- -glucal (4).—To a solution
D
of 3 (432 mg, 1.6 mmol) in dry DMF (12 mL)
at 23 °C was added imidazole (346 mg, 5.1

276
F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
green powder (1.25 g, 80%): 1H NMR
(CDCl3): l 8.05 (s, 2 H, HCN); 7.34 (dt, 2 H,
J 7.0, J 2 Hz, ArꢀH), 7.19 (dd, 2 H, J 8.0, J
1.5 Hz, ArꢀH), 7.12 (d, 2 H, J 8.5 Hz, ArꢀH),
6.66 (dt, 2 H, J 1.0, J 7 Hz, ArꢀH), 1.44 (s, 6
H, 2 CH3), 1.43 (s, 6 H, 2 CH3); 13C NMR
(CDCl3): l 168.05 (2 C, CꢁN), 162.93, 135.87,
134.14, 122.42, 120.60, 116.56 (12 C, ArꢀC),
72.58 (2 C, C(CH3)2), 25.66 (2 C, 2 CH3),
24.02 (2 C, 2 CH3); 15N NMR (CDCl3) l
1057.6 (Mnꢂ15N).
66.77 (minor), 63.162, 55.45, 55.20 (6 C, C-2,-
3,-4,-5,-6,OCH3), 25.73 (3 C, C(CH3)3), 18.19
(1 C, C(CH3)3), −3.81 (1 C, SiCH3), −4.94
(1 C, SiCH3); CIMS: m/z 508, [M+1]+.
Anal. Calcd for C22H32F3NO7Si: C, 52.06; H,
6.35; F, 11.23; N, 2.76; Si, 5.53. Found: C,
52.27; H, 6.26; F, 11.19; N, 2.48; Si, 5.29.
The procedure described above using (salt-
men)Mn(15N) yielded the corresponding 15N-
labeled lactol in identical yield (181 mg, 66%):
1H NMR (CDCl3): l 7.40 (m, 2 H, ArꢀH),
6.89 (m, 2 H, ArꢀH), 6.62 (broad dd, 1 H, JNH
90.9 Hz, 15NH (minor)), 6.46 (dd, 1 H, J 9.3,
3-O-tert-Butyldimethylsilyl-4,6-O-p-meth-
oxybenzylidene-2-deoxy-2-trifluoroacetamido-
15
D
-glucopyranose (5).—To a solution of 4 (235
JNH 91.8 Hz, NH (major)), 5.55–5.47 (4 s, 1
mg, 0.6 mmol) in CH2Cl2 (1 mL) and freshly
distilled trifluoroacetic anhydride (0.306 mL,
2.2 mmol) was added (saltmen)Mn(N) (10
mL, 0.06 M in CH2Cl2) with a syringe pump
during approximately 12 h (0.8 mL/h) at
23 °C and under Ar. After the addition, silica
gel (0.6 g), Celite® (0.6 g), and n-pentane (20
mL) were added, and the mixture was vigor-
ously stirred at 23 °C for 10 min. The mixture
was filtered, diluted in ice-cold satd aq
NaHCO3 (20 mL) and extracted. The organic
fraction was rapidly passed through a short
plug of silica gel with anhyd ether (150 mL)
and concentrated to give a yellow syrup. Silica
gel chromatography using a 10:90–33:67
EtOAc–hexanes gradient containing 0.1% of
Et3N yielded lactol 5 as a light-yellow foam
and as a mixture of diastereomers (210 mg,
66%): [h]D −30° (c 0.7, CHCl3); IR (thin
film): 3428 (OH), 2931, 1711 (CꢁO from
COCF3), 1615, 1519 (CꢁO from COCF3),
1382, 1303, 1251, 1169, 1129, 1083, 1047, 837,
H, CHꢀPMP), 5.28 (t, 1 H, J 3.3 Hz, H-1a
(gluco)), 4.91 (t, 1 H, J 8.1Hz, H-1b (gluco)),
4.41–3.99 (m, 4 H, H-2,-3,-4,-6a), 3.82 (s, 3 H,
OCH3), 3.76–3.46 (m, 2 H, H-5,-6b), 3.02
(broad s, 1 H, OH), 2.93 (d, J 4.2 Hz, OH),
0.85–0.79 (4 s, 9 H, C(CH3)3), 0.08 to −0.04
(6 s, 6 H, Si(CH3)2); 13C NMR (CDCl3): l
160.30 (1 C, CꢀOCH3 Ar), 129.67, 127.84,
127.81 (minor), 127.60 (minor), 113.70 (5 C, C
Ar), 102.24 (1 C, C acetal), 95.84 (minor),
92.14 (1 C, C-1), 82.46, 70.13, 68.95, 66.77
(minor), 63.05, 55.43 (5 C, C-3,-4,-5,-6,
1
OCH3), 55.30 (d, JC,N 10.5 Hz, C-2), 25.71 (3
C, C(CH3)3), 18.16 (1 C, C(CH3)3), −3.84 (1
C, SiCH3), −4.95 (1 C, SiCH3).
1-O-Acetyl-3-O-tert-butyldimethylsilyl-4,6-
O-p-methoxybenzylidene-2-deoxy-2-trifluoro-
acetamido- -glucopyranose (6).—To a solu-
D
tion of the lactol 5 (75 mg, 0.14 mmol) in
pyridine (3 mL) was added Ac2O (2 mL) at
0 °C under Ar, and the reaction was slowly
brought to 23 °C and stirred for 18 h. The
mixture was quenched at 0 °C with satd aq
NaHCO3 (30 mL), diluted in CHCl3 (60 mL),
extracted, dried over Na2SO4, and concen-
trated under reduced pressure to form a yel-
low foam. Silica-gel chromatography using a
10:90–25:75 EtOAc–hexanes gradient con-
taining 0.1% of Et3N afforded the pure gluco
a-acetate 6 (62 mg, 76%) and the remaining
three diastereomers (15 mg, 18%) as a mixture
of gluco b-acetate and manno a-/b-acetates in
1
781 cm−1; H NMR (CDCl3): l 7.40 (m, 2 H,
ArꢀH), 6.89 (m, 2 H, ArꢀH), 6.62 (broad d, 1
H, NH (minor)), 6.54 (d, 1 H, J 9.3 Hz, NH
(major)), 5.55–5.43 (4 s, 1 H, CHꢀPMP), 5.41
(s, 1 H, H-1 (minor)), 5.22 (d, 1 H, J1,2 3.6 Hz,
H-1a (gluco)), 5.00 (s, 1 H, H-1 (minor)), 4.85
(d, 1 H, J1,2 8.6Hz, H-1b (gluco)), 4.41–3.99
(m, 4 H, H-2,-3,-4,-6a), 3.81 (s, 3 H, OCH3),
3.76–3.40 (m, 2 H, H-5,-6b), 2.97–2.88 (2 s, 1
H, OH), 0.84–0.79 (4 s, 9 H, C(CH3)3), 0.08
to −0.04 (4 s, 6 H, Si(CH3)2); 13C NMR
(CDCl3): l 160.33 (1 C, CꢀOCH3 Ar), 129.71,
127.84, 127.60 (minor), 113.72 (5 C, C Ar),
102.27, 101.17 (minor) (1 C, C acetal), 95.84
(minor), 92.27 (1 C, C-1), 82.45, 70.13, 68.97,
1
a 63:37 ratio, as determined by H NMR
1
integration. H NMR (CDCl3) for 6: l 7.41
(m, 2 H, JA,A%=JX;X% 2.7, JA,X=JA%,X% 8.7 Hz,
ArꢀH), 6.91 (m, 2 H, JA,A%=JX;X% 2.7, JA,X
=
JA%,X% 8.7 Hz, ArꢀH), 6.19 (d, 1 H, J1,2 3.6 Hz,

F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
277
JX;X% 2.7, JA,X=JA%,X% 8.7 Hz, ArꢀH), 6.89 (m,
2 H, JA,A%=JX;X% 2.7, JA,X=JA%,X% 8.7 Hz,
ArꢀH), 6.48 (dd, 1 H, J 8.1, JNH 92.4 Hz,
15NH), 5.44 (s, 1 H, CHꢀPMP), 4.76 (dd, 1 H,
H-1), 6.14 (d, 1 H, J 8.7 Hz, NH), 5.50 (s, 1
H, CHꢀPMP), 4.36 (dt, 1 H, J2,3 9.3 Hz, H-2),
4.28 (dd, 1 H, J5,6a 4.5, J6a,6b 9.9 Hz, H-6a),
3.99 (t, 1 H, J3,4 9.3 Hz, H-3), 3.86 (dt, 1 H,
J4,5=J5,6b 9.9 Hz, H-5), 3.83 (s, 3 H, OCH3),
3.74 (t, 1 H, J6b,6a 10.2 Hz, H-6b), 3.61 (t, 1 H,
H-4), 2.21 (s, 3 H, COCH3), 0.82 (s, 9 H,
C(CH3)3), 0.05 (s, 3 H, SiCH3), 0.00 (s, 3 H,
SiCH3).
3
J1,2 8.4, J1,C 4.5 Hz, H-1), 4.34 (dd, 1 H, J5,6a
4.2, J6a,6b 10.2 Hz, H-6a), 4.21 (broad t, 1 H,
J 8.4 Hz, H-3), 3.82 (s, 3 H, OCH3), 3.77 (t, 1
H, H-6b), 3.58–3.42 (m, 3 H, H-2,-4,-5), 3.50
1
(d, 3 H, JC,H 143.7 Hz, O13CH3), 0.80 (s, 9 H,
Methyl 3-O-tert-butyldimethylsilyl-4,6-O-p-
methoxybenzylidene - 2 - deoxy - 2 - trifluoroace-
C(CH3)3), −0.01 (s, 3 H, SiCH3), −0.04 (s, 3
H, SiCH3); 13C NMR (CDCl3) (coupled): l
3
1
tamido-i-
D
-glucopyranoside
(8).—To
a
57.52 (dq, 1 C, JC,H1 4.5, JC,H 143.7 Hz,
O13CH3).
solution of the lactol 5 (45 mg, 0.09 mmol)
and silver(I) oxide (51 mg, 0.22 mmol) in dry
THF (1 mL) at 23 °C and under an Ar atmo-
sphere was added iodomethane (82 mL, 1.3
mmol). The mixture was allowed to stir for 5
days in a well-sealed flask then filtered over
Celite®, rinsed with EtOAc (40 mL), and con-
centrated. Silica gel chromatography using a
10:90–33:67 EtOAc–hexanes gradient con-
taining 0.1% of Et3N afforded compound 8 as
a white foam (35 mg, 75%): [h]D −37.8° (c
0.7, CHCl3); IR (thin film): 3305, 2931, 2858,
1704 (CꢁO from COCF3), 1616, 1560 (CꢁO
from COCF3), 1518, 1383, 1251, 1170, 1109,
Methyl 2-deoxy-2-trifluoroacetamido-i- -
D
glucopyranoside (9).—To a solution of com-
pound 8 (32 mg, 0.06 mmol) in dry THF was
added tetrabutylammonium fluoride (0.153
mL, 0.153 mmol) (1 M solution in THF). The
reaction was stirred under Ar at 23 °C for 18
h, cooled to 0 °C, diluted with satd aq
NaHCO3 (10 mL), and extracted twice with
EtOAc (2×15 mL). The combined organic
fractions were dried over Na2SO4, concen-
trated, and eluted with a short plug of silica
gel with EtOAc (100 mL) containing 0.1% of
Et3N to afford a white solid. This white solid
was dissolved in 8:1:1 HOAc–THF–water (5
mL) and stirred at 45 °C for 1.5 h. The mix-
ture was concentrated under reduced pressure,
and the resulting residue dissolved in warm
THF (ꢀ1 mL), transferred into a glass cen-
trifuge tube, and triturated with distilled hex-
anes (5 mL) to obtain a white precipitate. This
precipitate was centrifuged, washed with dis-
tilled hexanes (5 mL), and dried to afford a
white powder (16 mg, 90% after two steps):
mp 208–212 °C; [h]D −29° (c 1, CH3OH); IR
(thin film): 3280, 2940, 1707 and 1562 (CꢁO
from COCF3), 1210, 1186, 1159, 1075, 1032,
1
1094, 1076, 1036, 1006, 838, 781 cm−1; H
NMR (CDCl3): l 7.39 (m, 2 H, JA,A%=JX;X%
2.7, JA,X=JA%,X% 8.7 Hz, ArꢀH), 6.89 (m, 2 H,
JA,A%=JX;X% 2.7, JA,X=JA%,X% 8.7 Hz, ArꢀH),
6.37 (d, 1 H, J 8.1 Hz, NH), 5.46 (s, 1 H,
CHꢀPMP), 4.79 (d, 1 H, J1,2 8.4 Hz, H-1),
4.34 (dd, 1 H, J5,6a 4.2, J6a,6b 10.2 Hz, H-6a),
4.23 (dd, 1 H, J2,3 8.1 Hz, H-3), 3.82 (s, 3 H;
OCH3), 3.77 (t, 1 H, J5,6b 10.5 Hz, H-6b),
3.54–3.42 (m, 6 H, H-2,-4,-5 OCH3), 0.807 (s,
9 H, C(CH3)3), −0.02 (s, 3 H, SiCH3), −0.04
13
(s, 3 H, SiCH3); C NMR (CDCl3): l 160.32
(1 C, CꢀOCH3 Ar), 129.73, 127.84, 113.72 (5
C, C Ar), 102.09 (1 C, C acetal), 101.16 (1 C,
C-1), 82.38, 71.29, 68.76, 66.37, 59.49, 57.46,
55.45 (7 C, C-2,-3,-4,-5,-6, 2OCH3), 25.83 (3
C, C(CH3)3), 18.23 (1 C, C(CH3)3), −3.78 (1
C, SiCH3), −5.01 (1 C, SiCH3); CIMS: m/z
1
882, 634 cm−1; H NMR (CD3OD): l 4.41 (d,
1 H, J1,2 8.4 Hz, H-1), 3.91 (dd, 1 H, J 1.8, J
12.0 Hz, H-6a), 3.75–3.67 (m, 2 H, H-2,-6b),
3.54 (dd, 1 H, J2,3 7.8 Hz, H-3), 3.49 (s, 3 H,
13
522,
[M+H]+.
Anal.
Calcd
for
OCH3), 3.38–3.27 (m, 2 H, H-4,-5); C NMR
(CD3OD): 103.06 (1 C, C-1), 78.24 (1 C, C-5),
75.42 (1 C, C-4), 72.28 (1 C, C-3), 62.84 (1 C,
C-6), 57.80 (1 C, C-2), 57.26 (1 C, OCH3);
CIMS: m/z 290, [M+1]+. Anal. Calcd for
C9H14F3NO6: C, 37.38; H, 4.88; N, 4.84.
Found: C, 36.99; H, 4.94; N, 4.42.
C23H34F3NO7Si: C, 52.96; H, 6.57; N, 2.69.
Found: C, 53.11; H, 6.59; N, 2.55.
The procedure described above using 15N-
labeled lactol yielded the corresponding dou-
bly labeled methyl glucoside 8 (101 mg, 76%):
1H NMR (CDCl3): l 7.39 (m, 2 H, JA,A%
=

278
F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
Hz, H-1), 3.88 (dd, 1 H, J5,6a 2.1, J6a,6b 12 Hz,
The procedure described above using
13C,15N-labeled methyl glucoside 8 yielded the
H-6a), 3.68 (dd, 1 H, J5,6b 5.1 Hz, H-6b), 3.63
(dd, J2,3 9.9 Hz, H-2), 3.46 (s, 3 H, OCH3),
3.43 (t, 1 H, J3,4 10.5 Hz, H-3), 3.34–3.22 (m,
corresponding
doubly
labeled
N-trifl-
uoroacetylglucosamine as a white powder (21
13
1
2 H, H-4,-5), 1.97 (s, 3 H, COCH3); C NMR
mg, 94% after two steps): H NMR (CD3OD):
3
(D2O, referenced to Me4Si): l 174.92 (CꢁO),
102.16 (1 C, C-1), 76.11 (1 C, C-5), 74.18 (1 C,
C-3), 70.15 (1 C, C-4), 60.96 (1 C, C-6), 57.27
(1 C, C-2), 55.67 (1 C, OCH3), 22.36 (1 C,
COCH3); CIMS: m/z 236, [M+H]+. Anal.
Calcd for C9H17NO6: C, 45.95; H, 7.28; N,
5.95. Found: C, 45.69; H, 7.58; N, 5.61.
l 4.40 (d, 1 H, J1,C 4.5, J1,2 8.4 Hz, H-1), 3.91
(dd, 1 H, J 1.8, J 12.0 Hz, H-6a), 3.75–3.67
3
(m, 2 H, H-2,-6b), 3.54 (ddd, 1 H, JN,3 2.0,
1
J2,3 7.8 Hz, H-3), 3.47 (d, 3 H, JC,H 142.8 Hz,
O13CH3), 3.38–3.27 (m, 2 H, H-4,-5); 13C
NMR (CD3OD) (coupled): l 55.94 (dq, 1 C,
1
13
3JC,H1 4.5, JC,H 142.8 Hz, O13CH3); C NMR
1
2
(CD3OD): l 159.51 (dq, 1 C, JC,N 19.2, JC,F
The procedure described above using
13C,15N-labeled methyl glucoside 8 yielded the
corresponding doubly labeled N-acetylglu-
cosamine as a white powder (11 mg, 81% after
2
36.5 Hz, CꢁO), 117.76 (dq, 1 C, JC,N 11.1,
1JC,F 285.5 Hz, CF3), 103.06 (d, 1 C, JC1,C 2.2
2
Hz, C-1), 78.24 (s, 1 C, C-5), 75.42 (s, 1 C,
4
1
C-4), 72.28 (d, 1 C, JC3,C 1.6 Hz, C-3), 62.84
three steps): H NMR (CD3OD): l 4.31 (dd, 1
3
1
3
(s, 1 C, C-6), 57.80 (dd, 1 C, JC2,C 3.2, JC2,N
H, J1,2 8.4, J1,C 4.5 Hz, H-1), 3.88 (dd, 1 H,
10.2 Hz, C-2), 57.26 (s, 1 C, O13CH3); 15N
J5,6a 2.1, J6a,6b 12 Hz, H-6a), 3.68 (dd, 1 H,
1
2
NMR (CD3OD): l 113.7 (t, 1 N, JN,D 14 Hz,
J5,6b 5.1 Hz, H-6b), 3.63 (dt, JN,2 1.0, J2,3 9.9
1
15NTFA).
Hz, H-2), 3.46 (d, 3 H, JC,H 142.8 Hz,
3
Methyl 2-acetamido-2-deoxy-i-
D
-glucopy-
O13CH3), 3.43 (dt, 1 H, JN,3 1.5, J 10.5 Hz,
ranoside (10).—To a solution of compound 8
(42 mg, 0.08 mmol) in CH3OH (7 mL) and
water (1 mL) was added NaOH (0.8 g). The
reaction was brought to reflux for 7 h, cooled
to 23 °C, diluted with water (4 mL), and ex-
tracted several times with CHCl3 (4×10 mL).
The combined organic fractions were dried
over Na2SO4 and concentrated to give the
crude amine as a yellow solid. The amine was
redissolved in CH3OH (5 mL) at 0 °C and
treated with Ac2O (0.5 mL). The reaction was
stirred at 0 °C under Ar for 30 min, then
quenched at 0 °C with satd aq NaHCO3 (4
mL), and extracted several times with CHCl3
(3×15 mL). The combined organic fractions
were dried over Na2SO4, and concentrated to
afford a white solid. This white solid was
dissolved in 8:1:1 HOAc–THF–water (5 mL)
and stirred at 45 °C for 1.5 h. The solvents
were removed under reduced pressure, and the
residue was dissolved in hot THF (ꢀ1 mL),
transferred into a glass centrifuge tube and
triturated with distilled hexanes (5 mL) to
obtain a white precipitate. This precipitate
was centrifuged, washed with distilled hexanes
(5 mL), and dried to finally afford a white
powder (14 mg, 74% yield over three steps);
mp 194–196 °C; [h]D −42° (c 0.75, CH3OH);
1H NMR (CD3OD); l 4.31 (d, 1 H, J1,2 8.4
H-3), 3.34–3.22 (m, 2 H, H-4,-5), 1.97 (d, 3 H,
3JH,N 1.5 Hz, COCH3); 13C NMR (CD3OD)
3
1
(coupled): l 57.06 (dq, 3 C, JC,H1 4.5, JC,H
142.8 Hz, O13CH3); 13C NMR (CD3OD): l
173.94 (d, 1 C, 1JC,N 15.1 Hz, CꢁO), 103.72 (d,
2
1 C, JC1,C 2.3 Hz, C-1), 78.16 (s, 1 C, C-5),
4
76.40 (s, 1 C, C-4), 72.29 (d, 1 C, JC3,C 1.5
Hz, C-3), 62.94 (s, 1 C, C-6), 57.38 (dd, 1 C,
1
3JC2,C 3.2, JC2,N 10.6 Hz, C-2), 57.11 (s, 1 C,
2
O13CH3), 23.06 (d, 1 C, JC,N 8.3 Hz,
COCH3); 15N NMR (CD3OD): l 120.2 (t, 1
N, JN,D 14 Hz, 15NAc).
1
Methyl
2-azido-2-deoxy-i- -glucopyran-
D
oside (11).—To a solution of compound 8 (27
mg, 0.05 mmol) in CH3OH (7 mL), water (1
mL), was added NaOH (0.8 g). The mixture
was brought to reflux for 7 h, cooled to 23 °C,
diluted with water (5 mL), and extracted sev-
eral times with CHCl3 (4×10 mL). The com-
bined organic fractions were dried over
Na2SO4 and concentrated to afford the crude
amine as a yellow solid. The solid was redis-
solved at 0 °C with 90% CH3OH (7 mL), and
treated with the freshly made triflyl azide14
solution in CH2Cl2 (7 mL) (Note: TfN3 is
potentially explosive), CuSO4 (1 mg), and
K2CO3 (3 mg). The mixture was slowly
warmed up to 23 °C and stirred for 14 h under
Ar, carefully concentrated, and eluted with a

F.P. Boulineau, A. Wei / Carbohydrate Research 334 (2001) 271–279
279
15
(s, 1 C, O13CH3); N NMR (CD3OD): l 70.7
silica gel column using a 20:80–33:67 EtOAc–
hexanes gradient containing 0.1% of Et3N to
afford a white solid. This white solid was
dissolved in 8:1:1 HOAc–THF–water (5 mL)
and stirred at 45 °C for 1.5 h. The reaction
mixture was concentrated under reduced pres-
sure to afford a clear oil, which was eluted
with a short silica gel column with 9:1
CH2Cl2–CH3OH to afford the desired com-
pound as a solid (8.7 mg, 76% yield). This
solid can be crystallized from hexanes–THF
(see protocol for compounds 9 and 10): mp
111–113 °C; [h]D −22.8° (c 0.5, CH3OH); IR
(thin film): 3368, 2929, 2223, 2111 (N3), 1729,
1451, 1385, 1266, 1155, 1118, 1074, 1025, 955,
(s, 1 N, 15NN2).
Acknowledgements
The authors gratefully acknowledge finan-
cial assistance from the American Chemical
Society Petroleum Research Fund (36069-
AC1), the American Heart Association Mid-
west Affiliates (30399Z), and the Purdue
Research Foundation in the form of a fellow-
ship to F.P.B. The authors also wish to ac-
knowledge Dr H. Daniel Lee for providing
elemental analyses, Dr Karl V. Wood and
Arlene Rothwell for mass spectrometry ser-
vices, and Dr Klaas Hallenga and Dr Edwin
Rivera for NMR assistance.
1
892, 692 cm−1; H NMR (CD3OD): l 4.23 (d,
1 H, J1,2 8.1 Hz, H-1), 3.86 (dd, 1 H, J5,6a 2.1,
J6b,6a 12.0 Hz, H-6a), 3.68 (dd, 1 H, J5,6b 5.1,
H-6b), 3.55 (s, 3 H, OCH3), 3.32–3.24 (m, 3
H, H-3,-4,-5), 3.11 (t, 1 H, J2,3=J3,4 8.0 Hz,
H-2); 13C NMR (CD3OD): l 104.28 (1 C,
C-1), 78.09 (1 C, C-5), 76.57 (1 C, C-4), 71.70
(1 C, C-3), 68.38 (1 C, C-2), 62.67 (1 C, C-6),
57.41 (1 C, OCH3); CIMS: m/z 220, [M+1]+.
Anal. Calcd for C7H13N3O5: C, 38.36; H, 5.98;
N, 19.17. Found: C, 38.27; H, 5.74; N, 18.95.
The procedure described above using
13C,15N-labeled methyl glucoside 8 yielded the
corresponding doubly labeled 2-azido-2-de-
oxyglucose as a white crystalline solid (8.6 mg,
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1
73% after three steps): H NMR (CD3OD): l
3
4.23 (dd, 1 H, J1,C 4.5, J1,2 8.1 Hz, H-1), 3.86
(dd, 1 H, J5,6a 2.1, J6b,6a 12.0 Hz, H-6a), 3.68
(dd, 1 H, J5,6b 5.1 Hz, H-6b), 3.55 (d, 3 H,
1JC,H 142.2 Hz, O13CH3), 3.32–3.24 (m, 3 H,
H-3,-4,-5), 3.11 (t, 1 H, J2,3=J3,4 8.0 Hz, H-2);
13C NMR (CD3OD) (decoupled): l 57.25 (dq,
3
1
1 C, JC,H1 4.5, JC,H 142.2 Hz, O13CH3); 13C
2
NMR (CD3OD): l 104.28 (t, 1 C, JC1,C
=
2JC1,N 2.5 Hz, C-1), 78.09 (s, 1 C, C-5), 76.57
3
(d, 1 C, JC4,N 3.1 Hz, C-4), 71.70 (d, 1 C,
2JC3,N 2.2 Hz, C-3), 68.38 (t, 1 C, JC2,C
=
3
1JC2,N 3.2 Hz, C-2), 62.67 (s, 1 C, C-6), 57.41
.